P588: A MULTIPARAMETER FLOW CYTOMETRY PANEL FOR SIMPLE AND RAPID DIAGNOSIS OR SCREENING OF MALIGNANT TUMORS AND RELATED DISEASES
Authors: Hui Wang
Journal: HemaSphere (2023)

Background: Multiparameter flow cytometry (MFC) is the main technique for detecting acute leukemia, lymphoma, solid tumor, chronic myeloid neoplasm, and other malignant diseases. In addition, MFC can also detect pre-leukemia diseases, monoclonal B-cell lymphocytosis (MBL) and monoclonal gammopathy of undetermined significance (MGUS), and abnormalities of myeloid and lymphocyte caused by virus infections, immune, and genetic abnormalities, such as Paroxysmal Nocturnal Hemoglobinuria (PNH), aplastic anemia(AA), FCγ receptor IIIβ deficiency, Hemophagocytic lymphohistiocytosis (HLH), and primary immunodeficiency diseases (PIDs). Currently, various detection panels hinder the development of pre-processing equipment, analysis templates, and artificial intelligence. The existing simple guide tube method, which relies on clinical and morphological information may miss the minor clone. Aims: We developed a highly efficient panel screening or diagnosing most diseases, which could improve efficiency, prevent missed diagnoses, and promote standardized and intelligent development. Methods: We designed a 7-9 color, five-tube, one-step protocol for screening and diagnosing the vast majority of clonal diseases. The biomarkers in each tube were shown below: Tube1: CD7 FITC/CD117 PE/CD3 PerCP/CD4 Pecy7/CD5 APC/CD56 BV421/CD8 APCCy7/CD45 V500/CD2 BV605. Tube2: ckappa FITC/clambda PE/CD38 PerCP/CD19 Pecy7/CD10 APC/CD20 APCCy7/CD56 BV421/CD45 V500/CD5 BV605. Tube3：CD16 FITC/CD117 PE/CD34 PerCP/CD33 APC/CD13 PECy7/CD11b BV421/HLA-DR APCCy7/CD45 V500. Tube 4：MPO FITC/CD64 PE/CD34 PerCP/CD42a APC/CD36 APCCy7/CD45 V500/CD14 BV421. Tube5：TdT FITC/CD22 PE/CD34 PerCP/cCD3 APC/CD117 PECy7/CD38 BV421/CD45 V500. We used a 3-laser 10-color BD FACS cantoplus MFC and Diva software for multi-parameter gate analysis. From September 2020 to June 2021, we tested 4995 cases (2690 males and 2305 females) with a median age of 48 (0-94) years. The samples included 4893 bone marrow (BM) and 102 peripheral blood (PB). We identified 2455 positive cases, including 655 MDS, MPN, or MDS/MPN; 180 AA; 653 AML; 51 T-ALL/LBL; 267 B-ALL/LBL; 41 MPAL; 149 MM; 293 B-CLPD or MBL; 72 T-CLPD; 36 NK-CLPD; 15 solid tumors; 7 FCγ receptor IIIβ deficiency; 12 HLH with abnormal T or NK cells; 22 PNH; and 2 BPDCN. We validated our finding using morphological, genetic, and clinical manifestations, and a median follow-up of 5 months (1-12 months). Results: The sensitivity was 10-4, and the coverage and specificity were close to 100%. The false positive rate and false negative rate were nearly 0%. Only one case of CD56 negative epithelial-derived solid tumor could not be covered by the panel, but tumor cells were identified by CD45-/CD36-, with a proportion of 10%. Although additional antibodies are required for some diseases, such as mature T cell and NK cell neoplasms, solid tumors, and some rare diseases (including BPDCN, acute leukemia of ambiguity lineage, or mastocytosis), the new panel required 23% fewer antibodies (from an average of 6.5% on the basis of 40 antibodies in the previous method to 5%) than the previous Euroflow-like guide tube plus 5-6 determination tubes. To test the same amount of specimens, 33% of manpower was saved, and the probability of requiring additional testing after a two-step analysis was reduced from 5-10% to less than 1% for staff with 5-8 years of experience. Summary/Conclusion: Our comprehensive MFC diagnosis or screening panel minimizes the rate of missed diagnosis, improves efficiency, and is cost-efficient, thereby promotingautomation and standardization.Keywords: Immunophenotype, Acute leukemia, Flow cytometry